A. Cancer
One of the least understood and most complex disease processes is the transformation that occurs as a cell becomes malignant. This process involves both genetic mutations and proteomic transformations, the result of which allows the cell to escape normal controls preventing inappropriate cell division. All cancers are unique and distinct from other cells, as well as other cancers. Despite this uniqueness, cancer cells share some common attributes. Most cancer cells proliferate outside of the normal cell cycle controls, exhibit morphological changes and exhibit various biochemical disruptions to cellular processes.
Cancer is usually diagnosed when a tumor becomes visible well after the first on-set of cellular changes. Many cancers are diagnosed after a biopsy sample is examined by histology for morphologic abnormalities, evidence of cell proliferation and genetic irregularities. There is a clear need to identify and characterize new markers for malignancy. Recently there has been an effort to define markers for the diagnosis and prognosis of malignancies. Many of the genetic and biochemical changes occur during the early development of a tumor and these changes should be exploited for the early diagnosis of cancer.
Breast cancer is the leading cause of death among women in the Western world. Recent data suggests that there is a strong correlation between late detection and poor prognosis of this disease. Analysis of a thousand clinical cases indicates that there is extensive genetic damage and a high rate of DNA synthesis in breast tumors in comparison with normal breast tissue. These data suggest that an alteration in the DNA replication machinery of breast cancer cells may contribute to uncontrolled and error-prone DNA synthesis.
Human breast cells mediate DNA synthesis using the multiprotein replication complex termed the DNA synthesome (Coll et al, Oncology Research, 8:435-447 (1996)). The DNA synthesome is fully competent to support in vitro DNA replication. The transformation of non-malignant human breast cells to a malignant state is accompanied by an alteration to a specific component of DNA synthesome, Proliferating Cell Nuclear Antigen (PCNA). PCNA is a well-known cell-cycle marker protein, originally identified as an antigen for autoimmune disease (Bechtel et al, Cancer Research, 58:3264-3269 (1998)).
B. PCNA in Cancer/other Cell Processes
PCNA is currently used in the diagnosis of malignancy, as well as in evaluating the prognosis of the patient (Schonborn et al, J. Cancer Res. Clinical Oncology, 121:122 (1995)). PCNA is a small (36 kD) nuclear protein involved in many cellular processes. PCNA plays crucial roles in both DNA replication and DNA repair mechanisms. PCNA has also been associated with transcription events. PCNA forms a trimer in the nucleus and acts as an accessory protein to polymerase δ, and also interacts with a variety of other proteins (Downey et al, Cancer Cells, 6:1211-1218 (1988)). In the evaluation of malignancy, PCNA is often used as a marker for cell proliferation. However, PCNA alone does not correlate with the stage of malignancy or the patient outcome.
A novel PCNA from breast cancer cells has been identified. The malignant breast cancer cells express a unique, acidic form of PCNA protein, i.e., csPCNA, which can clearly be distinguished from the basic form of this protein found in non-malignant cells, i.e., nPCNA. This alteration is most likely the result of a post-translational modification (Bechtel et al, Cancer Res., 58:3264-3269 (1998)). However, prior to the present invention an effective method to purify csPCNA has not been described.
Recent advances in biochemical and genetic studies strongly indicate that PCNA may interact with different proteins involved in DNA mismatch repair, Okazaki fragments ligation, DNA methylation and chromatin assembly (Balajee et al, Mutat. Res., 404:3-11 (1998); Ceccotti et al, Curr Biol: 6:1528-1531 (1996); Chen et al, Proc. Natl. Acad. Sci., USA, 93:11597-11602 (1996); Chuang et al, Science, 277:1996-2000 (1997); Dimitrova et al, J. Cell. Biol., 146:709-722 (1999); Eki et al, J. Biol. Chem., 266:3087-3100 (1991); Eki et al, J. Biol. Chem., 267:7284-7294 (1992); Greene et al, Hum Mol Genet: 8, 2263-2273 (1999); Gu et al, Nucleic Acids Res., 26:1173-1178 (1998); Henderson et al, Embo J., 13:1450-1459 (1994); Johnson et al, J. Biol. Chem., 271:27987-27990 (1996); Kelman, Oncogene, 14:629-640 (1997); Kolodner et al, Curr. Opin. Genet. Dev., 9:89-96 (1999); Krude, Curr. Biol., 9:R394-R396 (1999); Lee et al, J. Biol. Chem., 266:22707-22717 (1991); Levin et al, Proc. Natl. Acad. Sci., USA, 94:12863-12868 (1997); Levin et al, Curr. Biol., 10:919-922 (2000); Martini et al, J. Cell. Biol., 143:563-575 (1998); Merrill et al, Genetics, 148:611-624 (1998); Mimura et al, Genes Cells, 5:439-452 (2000); Miura, J. Radiat. Res. (Tokyo), 40:1-12 (1999); Moggs et al, Mol. Cell. Biol., 20:1206-1218 (2000); Nishikawa et al, Jpn. J. Cancer. Res., 88:1137-1142 (1997); Otterlei et al, Embo J., 18:3834-3844 (1999); Pan et al, Proc. Natl. Acad. Sci., USA, 90:6-10 (1993); Schweitzer et al, Genetics, 152:953-963 (1999); Shibahara et al, Cell, 96:575-85 (1999); Sinicrope et al, Clin. Cancer. Res., 4:1251-1261 (1998); Tom et al, J. Biol. Chem., 276:24817-24825 (2001); Tomkinson et al, Mutat. Res., 407:1-9 (1998); Tsurimoto, Front. Biosci., 4:D849-D858 (1999); Umar et al, Cell, 87:65-73 (1996); and Wu et al, Nucleic Acids Res., 24:2036-2043 (1996)).
Xeroderma Pigmentosum (XP)G protein is reported to interact with PCNA (Gary et al, J. Biol. Chem., 272(39):24522-24529 (1997)). The DNA repair endonuclease XPG binds to proliferating cell nuclear antigen (PCNA) and shares sequence elements with the PCNA-binding regions of FEN-1 and cyclin-dependent kinase inhibitor p21. XPG is a repair endonuclease similar to FEN-1 and required for nucleotide excision repair. The human XPG endonuclease cuts on the 3′ since of a DNA lesion, during nucleotide excision repair.
In the present invention, XPG protein was unexpectedly found to be useful in selectively purifying csPCNA, and as a part of an ELISA system which can distinguish the csPCNA from the nPCNA. The detection of csPCNA in the ELISA serves as a powerful marker for early detection of malignancy.